The present invention provides a genus of compounds and pharmaceutical compositions that are protective for mitigating damage associated with tissue ischemia, particularly stroke (CNS ischemia), and ischemia of the myocardium. The present invention further provides a method for treating or preventing tissue damage precipitated by injury, disease or insult, particularly the tissue damage caused by ischemia. Lastly, the present invention provides a method for treating or preventing tissue damage by providing compounds that and compositions that inhibit or neutralize the cytotoxic activity of 3-aminopropanal.
Cerebral ischemia, a leading cause of disability and mortality world-wide, is mediated by a cascade of molecular cytotoxins that kill potentially viable cells in the brain. The polyamines, spemine, spermidine, and putrescine, which are among the most abundant molecules in mammalian brain, have been implicated in the pathogenesis of ischemic brain damage (Zhang et al., Proc. Natl. Acad. Sci. USA 91:10883-10887, 1994; Harman and Shaw, Br. J. Pharmac. 73:165-174, 1981; Bergeron et al., J. Med. Chem. 39:5257-5266, 1996; Glantz et al., J. Basic. Clin. Physiol. Pharmacol. 7:1-10, 1996; Dempsey et al., Neurosurg. 17:635-640, 1985; and Schmitz et al., Neurosurg. 33:882-888, 1993). Polyamine biosynthesis is increased following the onset of cerebral ischemia, due to an ischemia-mediated induction of ornithine decarboxylase, a key synthetic enzyme in the polyamine biosynthetic pathway. Spermine was linked to development of glutamate-mediated cytotoxicity, because it can bind to the NR1 subunit of the NMDA receptor and potentiate glutamate-mediated cell damage (Traynelis et al., Science 268:873-876, 1995; Traynelis and Cull-Candy. J. Physiol. (Lond.) 433:727-763, 1991; and Sullivan et al., Neuron 13:929-936, 1994). Administration of experimental therapeutics which inhibit ornithine decarboxylase limit the development of ischemic brain damage in experimental animal models of stroke [ref]. Thus, the accumulation of polyamines in the ischemic brain occupies an important role in the pathogenesis of stroke (Kindy et al., J. Cereb. Blood Flow Metab. 14:1040-1045, 1994).
Brain spermine and spermidine levels are actually decreased by cerebral ischemia (Paschen, J. Neurochem. 49:35-37, 1987; and Paschen, Cerebrovasc. Brain Metab. Rev. 4:59-88, 1992). This observed decline of tissue spermine and spermidine levels is accompanied by an increase in brain levels of putrescine (Paschen, Mol. Chem. Neuropathol. 16:241-271, 1992; Paschen, Cerebrovasc. Brain Metab. Rev. 4:59-88, 1992; Morgan, Bachrach and Heimer, eds. CRC Publications, 203-229, 1989; and Paschen et al., Acta Neuropathol. 76:388-394, 1988). Further, intracerebral putrescine levels correlated significantly with the volume of brain cell death. Putrescine does not interact with the NMDA receptor, and does not potentiate its cytotoxic activity. A possible explanation for these results may reside in the catabolism of polyamines via the xe2x80x9cinterconversion pathwayxe2x80x9d which is dependent upon the activity of tissue polyamine oxidase (Seiler and Bolkenius, Neurochem. Res. 10:529-544, 1985; Seiler et al., Med. Biol. 59:334-346, 1981; Bolkenius and Seiler, Int. J. Dev. Neurosci. 4:217-224, 1986; and Bolkenius et al., Biochim. Biophys. Acta 838:69-76, 1985). This ubiquitous enzyme, which is present in high levels in brain and other mammalian tissues, cleaves spermine and spermidine via oxidative deamination to generate the end products putrescine and 3-aminopropanal (Seiler and Bolkenius, Neurochem. Res. 10:529-544, 1985; Seiler, In Yu et al., eds. Elsevier Science, 333-344, 1995; Morgan, Essays in Biochemistry 23:82-115, 1987; and Houen et al., Acta Chem. Scand. 48:52-60, 1994). 3-Aminopropanal is known for its cytotoxicity to primary endothelial cells, fibroblasts, and a variety of transformed mammalian cell lines (Bouzyk and Rosiek, Cancer Lett. 39:93-99, 1988; Brunton et al., Toxic. in Vitro 8:337-341, 1994; Gaugas and Dewey, Br. J. Cancer 39:548-557, 1978; Morgan et al., J. Biochem. 236:97-101, 1986; and Ferrante et al., J. Immunol. 133:2157-2162, 1984). 3-Aminopropanal has also been implicated as a mediator of programmed cell death in murine embryonic limb buds, and may contribute to the development of necrosis in some tumors (Parchment and Pierce, Cancer Res 49:6680-6686, 1989; and Kurihara et al., Neurosurg. 32:372-375, 1993). Inhibition of polyamine oxidase with aminoguanidine blocked generation of 3-aminopropanal in cell cultures following the addition of spermine, and prevented subsequent cytotoxicity (Ferrante et al., J. Immunol. 133:2157-2162, 1984; Morgan, Essays in Biochemistry 23:82-115, 1987; and Parchment and Pierce, Cancer Res. 49:6680-6686, 1989). On a molar basis, the LD50 concentration of 3-aminopropanal to cells is similar to the cytotoxicity of glutamate. In contrast, putrescine is not cytotoxic to cells, even in the millimolar range, but its rate of production through polyamine oxidation correlates directly with the formation of a directly cytotoxic aldehyde, 3-aminopropanal.
In addition, in the data first being reported herein in glial cells, 3-aminopropanal mediates apoptosis by activation of an interleukin-1 beta converting enzyme (ICE)-dependent signaling pathway, whereas in neurons it causes necrotic cell death.
Cerebral ischemia (stroke) is a debilitating condition resulting from a sudden cessation of blood flow to an area of the brain, resulting in a loss of brain tissue. There are no available therapies to reverse the neurological deficits caused by neuronal death in the infarct zone. Stroke is a major public health problem in the United States wherein about 550,000 strokes occur each year. Cerebral ischemia afflicts individuals of all age groups, but the incidence doubles with each decade over 45 and reaches 1-2% per year in the population of individuals over 75 years of age. If a patient survives, major disability can result with loss of ability to communicate, ambulate, see, coordinate and/or reason. Standard therapy is often ineffective at preventing brain infarction and is meant to support cardiovascular and respiratory function, control intracranial pressure, and prevent recurrent stroke. There is also a class of protease enzymes that are designed to dissolve blood clots, only for those strokes caused by blood clots potentially useful in brain ischemia but (as opposed to bleeding) and these agents only function to restore some blood flow in limited situations.
During the evolution of cerebral infarction (stroke), a core of densely ischemic tissue becomes rapidly and irreversibly damaged. Cellular damage in the surrounding area, termed the xe2x80x9cischemic penumbra,xe2x80x9d progresses more slowly.
Following an ischemic insult, the process of tissue destruction may not be completed for hours or even days (Kirino et al., Acta Neuropathol. 64:139-147, 1984; and Petito et al. Neurology 37:1281-1286, 1987). There is a temporary window of opportunity for an intervention to prevent ischemic tissue from progressing to infarction. In humans, this window is thought to extend from about 2-4 hours following the onset of ischemia, after which time the efficacy decreases rapidly (Ginsberg and Pulsinelli, Ann. Neurol. 36:553-554, 1994). During the therapeutic window, the target for therapeutic neuroprotection is the ischemic penumbra, a volume of brain tissue around the ischaemic core, which receives reduced blood flow and contains compromised, but potentially viable tissue. Studies have identified important cytotoxic mediators that cause cell death in the early hours after the onset of ischemia.
A number of molecular substrates of normal brain, as well as extrinsic factors delivered by the circulation, contribute to the development of cell cytotoxicity during ischemia. These include, but are not limited to, glutamate, aspartate, nitric oxide, calcium, free radicals, zinc, cytokines, arachidonic acid metabolites, and advanced glycation end products (AGEs). Advanced glycation endproducts are a group of protein modifying adducts that were implicated in the pathogenesis of diabetic complications. AGEs were found to be cerebrotoxic in the ischemic penumbra (Zimmerman et al., Proc. Natl. Acad. Sci. USA 92:3744-3748, 1995). In addition, aminoguanidine, a small molecule inhibitor of AGE cross-linking reactions, effectively abrogated the cerebrotoxicity of AGEs during focal cerebral ischemia (Zimmerman et al., Proc. Natl. Acad. Sci. USA 92:3744-3748, 1995). Aminoguanidine was also found to be cerebroprotective during focal ischemia in normal, non-diabetic animals, independent of exogenous AGEs (Zimmerman et al., Surg. Forum. 45:600-603, 1994). Aminoguanidine further provided cerebroprotection in a model of focal stroke when administered within 2 hours after the onset of focal cerebral ischemia (Cockroft et al., Stroke 27:1393-1398, 1996). It was considered that the mechanism of action was inhibition of polyamine oxidase (PAO), an enzyme that produces toxic, reactive aldehyde metabolites by oxidation of biogenic amines.
The cascade of cytotoxicity that is initiated by reduced blood flow is followed by a drop in ATP levels and a reduction of oxidative phosphorylation. As a result, membrane potentials fall, leading to release of K+ and an excessive amount of glutamate and other excitatory amino acids (EAAs) in a process called excitotoxicity. This will, in turn, over-activate N-methyl-D-aspartate (NMDA), amino-3-hydroxy-5-methyl-4-isoxasole-4-propionate (AMPA), kainate (KA), and 1S,3R-trans-1-amino-cyclopentyl-1,3dicarboxylate (trans-ACPD) receptors (Faroquil and Horrocks, Brain Res. 16:171-191, 1991).
Elevated glutamate leads to excessive Ca2+ influx, primarily by excitatory amino acid receptor channel activation, as well as swelling and osmotic lysis as a result of depolorization mediated influx of Na+, Clxe2x88x92 and water (Faroquil and Horrocks, Brain Res. 16:171-191, 1991). This elevation of intracellular Ca2+ activates phospholipases, lipases, proteases and protein kinases, leading to eventual breakdown of phospholipid membranes, cytoskeletal alterations, arachidonic acid release, and potentiation of the free radical cascade (Manfred et al., Biochem. Pharm. 50:1-16, 1995). Other modulators of NMDA receptors include Zn2+, histamine, certain neuroactive steroids, arachidonic acid, polyamines and protons or pH (Collinridge and Lester, Pharmacol. Rev. 74:143-210, 1989; and McBain and Mayer, Physiol. Rev. 74:723-760, 1994). Moreover, an NMDA receptor antagonist, MK-801, can exert a neuroprotective effect in animal models of cerebral ischemia (Olney et al., J. Neurosci. 9:1701-1704, 1989).
Ischemia also leads to formation of reactive oxygen species (ROS), activation of lipid peroxidation, and a reduction in the endogenous antioxidants ascorbate, glutathione, ubiquinone and xcex1-tocopherol in brain tissue. The mitochondrial respiratory chain and reaction sequences catalyzed by cyclooxygenase and lipoxygenase are important production sites for superoxide anion (O2xe2x88x92), hydrogen peroxide (H2O2) and hydroxy radical (OHxe2x88x92). Activated oxygen species are also formed during autooxidation of catecholamines and in the xanthine reaction.
Nitric oxide (NO) is another mediator of tissue injury in cerebral ischemia. NO concentrations increase acutely in the brain after middle cerebral artery (MCA) occlusion, from approximately 10 nM to 2.2 xcexcM by a porphyrinc microsensor assay (Beckman et al., Proc. Natl. Acad. Sci. USA 87:1620, 1990).
In addition to these other suspected mediators of ischemic tissue damage, 3-aminopropanol is an enzymatic by-product of the oxidative cleavage of the polyamines spermine and spermidine by PAO in mammalian cells (Holtta, Biochemistry 16:91-100, 1997). The cytotoxicity resulting from co-incubation of PAO activity with spermine and spermidine has been abolished by aminoguanidine (Gabl et al., Chemicobiological Interactins 22:91-98, 1978; and Henle et al., Cancer Res. 46:175-182, 1986). 3-Aminopropanal has also been implicated in causing programmed cell death in murine embryonic limits buds (Parchment et al., Cancer Res. Arch. 49:6680-6686, 1989) and in necrosis of solid tumors.
These data provides a need in the art to find inhibitors of PAO activity that are likely to have therapeutic utility in treating tissue ischemia, particularly mitigating damage to the ischemic penumbra experienced in stroke, but also in non-neuronal tissue such as muscle tissue (e.g., smooth muscle and cardiac muscle).
The present invention provides a stroke-damage mitigating compound having a formula I: 
wherein R and R1 are independently hydrogen, sulfamide, carboxyamide, cyano, straight or branched C1-6 alkyl, straight or branched C2-6 alkenyl, straight or branched C1-6 alkoxy, a straight chain C1-6 alkyl or a straight chain C2-6 alkenyl having an ether link or an ester link, toluenyl, COOH, nitrate, or halide (Br, Cl, I, F), wherein both R and R1 cannot be hydrogen, wherein R2 and R3 are independently hydrogen, sulfamide, carboxyamide, cyano, straight or branched C1-6 alkyl, straight or branched C2-6 alkenyl, straight or branched C1-6 alkoxy, a straight chain C1-6 alkyl or a straight chain C2-6 alkenyl having an ether link or an ester link, toluenyl, COOH, nitrate, or halide (Br, Cl, I, F).
Preferably, R and R1 are meta to each other and to the heteroatom. Preferably, R is COOH. Preferably, R1 is COOH. Preferably, R2 and R3 are both hydrogen. Most preferably, R and R1 are each COOH, and R2 and R3 are both hydrogen.
Preferred compounds of formula I include, for example, 1-phenacyl-2,3-dicarboxypyridinium bromide; 1-phenacyl-2,4-dicarboxypyridinium bromide; 1-phenacyl-2,5-dicarboxypyridinium bromide; 1-phenacyl-2,6-dicarboxypyridinium bromide; 1-phenacyl-2,3-dicarboxyimidepyridinium bromide; 1-phenacyl-2,4-dicarboxyimidepyridinium bromide; 1-phenacyl-2,5-dicarboxyimidepyridinium bromide; and 1-phenacyl-2,6-dicarboxyimidepyridinium bromide; 1-phenacyl-2,3-dicarboxyimidepyrdinium bromide; 1-phenacyl-2,4-dicarboxyimidepyrdinium bromide; 1-phenacyl-2,5-dicarboxyimidepyrdinium bromide; and 1-phenacyl-2,6-dicarboxyimidepyrdinium bromide.
The present invention provides a pharmaceutical composition comprising a compound from formula I in a pharmaceutically acceptable carrier, wherein formula I comprises: 
wherein R and R1 are independently hydrogen, sulfamide, carboxyamide, cyano, straight or branched C1-6 alkyl, straight or branched C2-6 alkenyl, straight or branched C1-6 alkoxy, a straight chain C1-6 alkyl or a straight chain C2-6 alkenyl having an ether link or an ester link, toluenyl, COOH, nitrate, or halide (Br, Cl, I, F), wherein both R and R1 cannot be hydrogen, wherein R2 and R3 are independently hydrogen, sulfamide, carboxyamide, cyano, straight or branched C1-6 alkyl, straight or branched C2-6 alkenyl, straight or branched C1-6 alkoxy, a straight chain C1-6 alkyl or a straight chain C2-6 alkenyl having an ether link or an ester link, toluenyl, COOH, nitrate, or halide (Br, Cl, I, F).
Preferably, R and R1 are meta to each other and to the heteroatom. Preferably, R is COOH. Preferably, R1 is COOH. Preferably, R2 and R3 are both hydrogen. Most preferably, R and R1 are each COOH, R2 and R3 are both hydrogen.
The present invention further provides a method for treating tissue ischemia to mitigate ischemic damage, comprising administering an effective amount of a compound of formula I, wherein formula I comprises: 
wherein R and R1 are independently hydrogen, sulfamide, carboxyamide, cyano, straight or branched C1-6 alkyl, straight or branched C2-6 alkenyl, straight or branched C1-6 alkoxy, a straight chain C1-6 alkyl or a straight chain C2-6 alkenyl having an ether link or an ester link, toluenyl, COOH, nitrate, or halide (Br, Cl, I, F), wherein both R and R1 cannot be hydrogen, wherein R2 and R3 are independently hydrogen, sulfamide, carboxyamide, cyano, straight or branched C1-6 alkyl, straight or branched C2-6 alkenyl, straight or branched C1-6 alkoxy, a straight chain C1-6 alkyl or a straight chain C2-6 alkenyl having an ether link or an ester link, toluenyl, COOH, nitrate, or halide (Br, Cl, I, F).
Preferably, R and R1 are meta to each other and to the heteroatom. Preferably, R is COOH. Preferably, R1 is COOH. Preferably, R2 and R3 are both hydrogen. Most preferably, R and R1 are each COOH, and R2 and R3 are both hydrogen.
The invention further provides a method for treating tissue ischemia to mitigate ischemic damage, comprising administering an effective amount of a compound of formula II, wherein formula II comprises: 
wherein R1 and R2 are independently selected from the group consisting of hydrogen, hydroxy C1-6 alkyl, C1-6 alkoxy C1-6 alkyl, and R1 and R2 together with their ring carbons may be an aromatic fused ring; wherein Z is hydrogen or an amino group; wherein Y is hydrogen or a group of the formula xe2x80x94CH2COR; wherein R is C1-6 alkyl, C1-6 alkoxy, hydroxy, amino, aryl, or xe2x80x94CH2R3 wherein R3 is H, C1-6 alkyl, C2-6 alkenyl, or C4-6 aryl. Preferably, the compound of formula II is a halide (Cl, Br, F or I), tosylate, methanesulfonate or mesitylene sulfonate salt.
The present invention further provides a method for inhibiting tissue damage, comprising administering an effective amount of a compound that inhibits or neutralizes the cytotoxic activity of 3-aminopropanal. Preferably, the diseases resulting from tissue ischemia are myocardial infarction or stroke.